U.S. patent number 6,720,419 [Application Number 10/278,942] was granted by the patent office on 2004-04-13 for sulfated fucan oligosaccharide.
This patent grant is currently assigned to Takara Bio Inc.. Invention is credited to Hitomi Amarume, Katsushige Ikai, Ikunoshin Kato, Takashi Kawai, Kaoru Kojima, Takeshi Sakai, Kazuo Shimanaka.
United States Patent |
6,720,419 |
Sakai , et al. |
April 13, 2004 |
Sulfated fucan oligosaccharide
Abstract
A smaller molecule obtainable by allowing a sulfated
fucan-digesting enzyme which digests a novel sulfated
polysaccharide derived from an alga belonging to Laminariales to
act on a sulfated fucan, and a method for producing the same.
Inventors: |
Sakai; Takeshi (Shiga,
JP), Amarume; Hitomi (Aomori, JP), Kawai;
Takashi (Shiga, JP), Kojima; Kaoru (Aomori,
JP), Shimanaka; Kazuo (Osaka, JP), Ikai;
Katsushige (Shiga, JP), Kato; Ikunoshin (Kyoto,
JP) |
Assignee: |
Takara Bio Inc. (Otsu,
JP)
|
Family
ID: |
19142424 |
Appl.
No.: |
10/278,942 |
Filed: |
October 24, 2002 |
Foreign Application Priority Data
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Oct 24, 2001 [JP] |
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2001-325960 |
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Current U.S.
Class: |
536/123.1;
536/118; 536/122; 536/124; 536/4.1; 568/28; 536/18.5 |
Current CPC
Class: |
C08B
37/006 (20130101) |
Current International
Class: |
C08B
37/00 (20060101); C08B 037/00 (); C07H 005/10 ();
C07H 003/04 (); C07H 003/06 () |
Field of
Search: |
;536/55.1,123.1,4.1,122,118,124,18.5 ;568/28 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1 057 833 |
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Dec 2000 |
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EP |
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1 175 907 |
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Jan 2002 |
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EP |
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1 176 153 |
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Jan 2002 |
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EP |
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WO 90/15823 |
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Dec 1990 |
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WO |
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WO 97/08206 |
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Mar 1997 |
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WO |
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WO 01/81560 |
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Nov 2001 |
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WO |
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Other References
Masato Nagaoka et al "Structural Study of Fucoidan from Cladosiphon
Okamuranus Tokida" Glycoconjugate Journal (1999) 16:19-26..
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Primary Examiner: Barts; Samuel
Assistant Examiner: Henry; Michael C.
Attorney, Agent or Firm: Browdy and Neimark, PLLC
Claims
What is claimed is:
1. A sulfated fucan having the following chemical and physical
properties: (1) containing fucose as a constituting saccharide; (2)
containing a sulfated saccharide of general formula (I) as an
essential component of the constituting saccharide: ##STR17##
wherein R is H or SO.sub.3 H, at least one of Rs is SO.sub.3 H and
n is an integer of 1 or more; and (3) being converted into smaller
molecules by a sulfated fucan-digesting enzyme derived from
Alteromonas sp. SN-1009 to generate at least one compound selected
from the group consisting of the compounds of general formulas
(II), (III), (XIII), (XIV), (XV) and (XVI): ##STR18## ##STR19##
##STR20##
wherein R is H or SO.sub.3 H and at least one of Rs is SO.sub.3 H
in all formulas above, or a salt thereof.
2. A sulfated fucan oligosaccharide of general formula (I):
##STR21##
wherein R is H or SO.sub.3 H, at least one of Rs is SO.sub.3 H and
n is 1 to 5.
3. A method for preparing a sulfated fucan oligosaccharide, the
method comprising: allowing a sulfated fucan-digesting enzyme
derived from Alteromonas sp. SN-1009 to act on a sulfated fucan
defined by claim 1 or 2; and collecting a digestion product.
4. The method according to claim 3, wherein the sulfated fucan is
derived from Kjellmaniella crassifolia, Laminaria japonica or
Lessonia nigrescens.
5. A sulfated fucan oligosaccharide having a chemical structure
selected from the group consisting of general formulas (II), (III),
(XIII), (XIV), (XV) and (XVI): ##STR22##
wherein R is H or SO.sub.3 H and at least one of Rs is SO.sub.3 H
in all formulas above, or a salt thereof.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a sulfated fucan oligosaccharide
which is useful in a field of glycotechnology, and a method for
producing the same.
2. Description of Related Art
Brown algae contain a variety of sulfated polysaccharides. These
polysaccharides are often generically called fucoidans or
fucoidins. In many cases, their structures vary depending on the
algae from which they derive. For example, sulfated polysaccharides
extracted from Fucus vesiculosus, Laminaria japonica, Cladosiphon
okamuranus, Nemacystus decipiens and sporophyll of Undaria
pinnatifida have structures different each other. Therefore, it is
necessary to obtain enzymes that digest the respective sulfated
polysaccharides in order to obtain oligosaccharides from the
sulfated polysaccharides by enzymatically digesting them or to
determine their structures.
Molecular species of sulfated polysaccharides including sulfated
fucans, sulfated fucoglucuronomannans and sulfated fucogalactans as
well as several other molecular species have been reported.
Sulfated polysaccharides generally have some biological activities
in many cases. For example, a sulfated fucan fraction has been
reported to have a strong anticoagulant activity, and a sulfated
fucoglucuronomannan fraction has been reported to have an
apoptosis-inducing activity against tumor cells.
If a sulfated polysaccharide is to be developed as a
pharmaceutical, it is necessary to determine its structure. It is
very advantageous to determine the structure using an enzyme that
digests the sulfated polysaccharide. However, no enzyme that
digests a sulfated polysaccharide from a brown alga is commercially
available. In addition, a digesting enzyme that specifically
digests the sulfated polysaccharide of which the structure is to be
determined is required. This is because sulfated polysaccharides
from brown algae vary depending on the species of the algae in many
cases. Structures of sulfated polysaccharides derived from algae
belonging to Laminariales have been studied, although structures
have been revealed only for a few kinds among many molecular
species.
As described above, a structurally homogeneous sulfated fucan
oligosaccharide which is produced by enzymatic means using an
enzyme that digests a novel sulfated polysaccharide derived from an
alga belonging to Laminariales (i.e., an enzyme that specifically
digests a sulfated fucan) has been desired.
Thus, the main object of the present invention is to provide a
smaller molecule obtainable by allowing a sulfated fucan-digesting
enzyme which digests a novel sulfated polysaccharide derived from
an alga belonging to Laminariales to act on a sulfated fucan, and a
method for producing the same.
SUMMARY OF THE INVENTION
The first aspect of the present invention relates to a sulfated
fucan having the following chemical and physical properties: (1)
containing fucose as a constituting saccharide; (2) containing a
sulfated saccharide of general formula (I) as an essential
component of the constituting saccharide: ##STR1##
wherein R is H or SO.sub.3 H, at least one of Rs is SO.sub.3 H and
n is an integer of 1 or more; and (3) being converted into smaller
molecules by a sulfated fucan-digesting enzyme derived from
Alteromonas sp. SN-1009 to generate at least one compound selected
from the group consisting of the compounds of general formulas
(II), (III), (XIII), (XIV), (XV) and (XVI): ##STR2##
wherein R is H or SO.sub.3 H and at least one of Rs is SO.sub.3 H
in all formulas above, or a salt thereof.
The second aspect of the present invention relates to a sulfated
fucan oligosaccharide of general formula (I): ##STR3##
wherein R is H or SO.sub.3 H, at least one of Rs is SO.sub.3 H and
n is 1 to 5.
The third aspect of the present invention relates to a method for
preparing a sulfated fucan oligosaccharide, the method comprising:
allowing a sulfated fucan-digesting enzyme derived from Alteromonas
sp. SN-1009 to act on a sulfated fucan of the first or second
aspect; and collecting a digestion product.
According to the third aspect, the sulfated fucan is derived from
Kjellmaniella crassifolia, Laminaria japonica or Lessonia
nigrescens.
The fourth aspect of the present invention relates to a sulfated
fucan oligosaccharide obtainable by the preparation method of the
third aspect.
The fifth aspect of the present invention relates to a sulfated
fucan oligosaccharide having a chemical structure selected from the
group consisting of general formulas (II), (III), (XIII), (XIV),
(XV) and (XVI): ##STR4##
wherein R is H or SO.sub.3 H and at least one of Rs is SO.sub.3 H
in all formulas above, or a salt thereof.
As a result of intensive study, the present inventors have found a
method for producing a sulfated fucan oligosaccharide by digesting
a novel sulfated polysaccharide derived from a alga belonging to
Laminariales using a sulfated fucan-digesting enzyme. The sulfated
fucan oligosaccharide can be utilized as a reagent for
glycotechnology and is structurally homogeneous. Furthermore, the
present inventors have determined the structure of the
oligosaccharide. Thus, the present invention has been
completed.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1: a figure which illustrates the .sup.1 H-NMR spectrum of the
sulfated fucan oligosaccharide 1-(1) according to the present
invention.
FIG. 2: a figure which illustrates the .sup.13 C-NMR spectrum of
the sulfated fucan oligosaccharide 1-(1) according to the present
invention.
FIG. 3: a figure which illustrates the mass spectrum of the
sulfated fucan oligosaccharide 1-(1) according to the present
invention.
FIG. 4: a figure which illustrates the .sup.1 H-NMR spectrum of the
sulfated fucan oligosaccharide 1-(2) according to the present
invention.
FIG. 5: a figure which illustrates the .sup.13 C-NMR spectrum of
the sulfated fucan oligosaccharide 1-(2) according to the present
invention.
FIG. 6: a figure which illustrates the mass spectrum of the
sulfated fucan oligosaccharide 1-(2) according to the present
invention.
FIG. 7: a figure which illustrates the .sup.1 H-NMR spectrum of the
sulfated fucan oligosaccharide 1-(3) according to the present
invention.
FIG. 8: a figure which illustrates the .sup.13 C-NMR spectrum of
the sulfated fucan oligosaccharide 1-(3) according to the present
invention.
FIG. 9: a figure which illustrates the mass spectrum of the
sulfated fucan oligosaccharide 1-(3) according to the present
invention.
FIG. 10: a figure which illustrates the .sup.1 H-NMR spectrum of
the sulfated fucan oligosaccharide 1-(4) according to the present
invention.
FIG. 11: a figure which illustrates the .sup.13 C-NMR spectrum of
the sulfated fucan oligosaccharide 1-(4) according to the present
invention.
FIG. 12: a figure which illustrates the mass spectrum of the
sulfated fucan oligosaccharide 1-(4) according to the present
invention.
FIG. 13: a figure which illustrates the .sup.1 H-NMR spectrum of
the sulfated fucan oligosaccharide 2-(1)-2 according to the present
invention.
FIG. 14: a figure which illustrates the .sup.13 C-NMR spectrum of
the sulfated fucan oligosaccharide 2-(1)-2 according to the present
invention.
FIG. 15: a figure which illustrates the mass spectrum of the
sulfated fucan oligosaccharide 2-(1)-2 according to the present
invention.
FIG. 16: a figure which illustrates the .sup.1 H-NMR spectrum of
the sulfated fucan oligosaccharide 2-(2) according to the present
invention.
FIG. 17: a figure which illustrates the .sup.13 C-NMR spectrum of
the sulfated fucan oligosaccharide 2-(2) according to the present
invention.
FIG. 18: a figure which illustrates the mass spectrum of the
sulfated fucan oligosaccharide 2-(2) according to the present
invention.
FIG. 19: a figure which illustrates the .sup.1 H-NMR spectrum of
the Lessonia nigrescens sulfated fucan oligosaccharide 1-(1)
according to the present invention.
FIG. 20: a figure which illustrates the .sup.13 C-NMR spectrum of
the Lessonia nigrescens sulfated fucan oligosaccharide 1-(1)
according to the present invention.
FIG. 21: a figure which illustrates the mass spectrum of the
Lessonia nigrescens sulfated fucan oligosaccharide 1-(1) according
to the present invention.
FIG. 22: a figure which illustrates the .sup.1 H-NMR spectrum of
the Lessonia nigrescens sulfated fucan oligosaccharide 1-(2)
according to the present invention.
FIG. 23: a figure which illustrates the .sup.13 C-NMR spectrum of
the Lessonia nigrescens sulfated fucan oligosaccharide 1-(2)
according to the present invention.
FIG. 24: a figure which illustrates the mass spectrum of the
Lessonia nigrescens sulfated fucan oligosaccharide 1-(2) according
to the present invention.
FIG. 25: a figure which illustrates the .sup.1 H-NMR spectrum of
the Lessonia nigrescens sulfated fucan oligosaccharide 1-(3)
according to the present invention.
FIG. 26: a figure which illustrates the .sup.13 C-NMR spectrum of
the Lessonia nigrescens sulfated fucan oligosaccharide 1-(3)
according to the present invention.
FIG. 27: a figure which illustrates the mass spectrum of the
Lessonia nigrescens sulfated fucan oligosaccharide 1-(3) according
to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be explained in detail.
Although it is not intended to limit the present invention, for
example, a sulfated fucan derived from an alga belonging to
Laminariales can be used according to the present invention. This
polysaccharide has a sulfate group and fucose as main constituents.
The main chain of a sulfated fucan derived from an alga belonging
to Laminariales is composed of L-fucose which is more acid-labile
than general saccharides. Therefore, it can be readily converted
into smaller molecules by heating or acid treatment.
A sulfated fucan having the above-mentioned characteristics can be
used according to the present invention. Although there is no
specific limitation concerning the origin, for example, algae
belonging to Laminariales such as Kjellmaniella crassifolia,
Laminaria japonica and Lessonia nigrescens are preferably used as
raw materials because their sulfated fucan contents are high.
The sulfated fucan-digesting enzyme according to the present
invention acts on a sulfated fucan, a sulfated fucan
oligosaccharide and the like and hydrolyzes an .alpha.-L-fucosyl
bond between fucoses in an endo-type manner to generate an
oligosaccharide having L-fucose at its reducing end.
The sulfated fucan oligosaccharide of the present invention is an
oligosaccharide that is obtainable by allowing the sulfated
fucan-digesting enzyme according to the present invention to act on
a sulfated fucan and has L-fucose as a saccharide at the reducing
end.
A water-soluble fraction extract is first obtained from a brown
alga in order to produce the sulfated fucan used according to the
present invention. In this case, it is preferable to obtain the
water-soluble fraction extract at pH 4-9 at a temperature of
100.degree. C. or below in order to prevent the conversion of the
sulfated fucan into smaller molecules. Furthermore, amino acids,
small molecule pigments or the like in the extract can be
efficiently removed by ultrafiltration. Activated carbon treatment
or the like is effective for the removal of hydrophobic
substances.
A sulfated polysaccharide fraction from a brown alga can be
obtained as described above. This fraction can be used as a
sulfated fucan fraction, for example, as a substrate for the
sulfated fucan-digesting enzyme according to the present invention.
A more highly pure sulfated fucan can be obtained by separating the
fraction using an anion exchange column. Both the sulfated
polysaccharide fraction and the sulfated fucan purified using an
anion exchange column can be used as raw materials for production
of the sulfated fucan oligosaccharide of the present invention.
The main backbone of the sulfated fucan of the present invention is
represented by general formula (I) below. The sulfated fucans of
the present invention include those of the general formula wherein
n is an integer of 1 or more, for example 1 to 20,000, preferably 1
to 10,000. The sulfated fucans of the present invention include
those having a structure in which general formula (I) is
continuously repeated and those having a structure in which general
formula (I) is discontinuously included being intervened by other
structures as long as they are within the definition as described
above. ##STR5##
wherein R is H or SO.sub.3 H, and at least one of Rs is SO.sub.3
H.;
The bacterial strain producing the sulfated fucan-digesting enzyme
to be used according to the present invention is classified as a
bacterium belonging to genus Alteromonas according to the basic
classification as described in Bergey's Manual of Determinative
Bacteriology, Vol. 9 (1994). Classification of bacteria belonging
to genus Alteromonas is recently re-organized. Therefore, it is not
appropriate to classify the bacterium based only on the
bacteriological properties. The nucleotide sequence of 16S rDNA of
this bacterial strain was determined. Comparison of homologies with
sequences of known bacteria revealed that a bacterium belonging to
genus Thalassomonas has the most homologous sequence. However,
since the genetic distance is 0.05 (change/average nucleotide
position) or more, the bacterium was not determined to belong to
this genus. Then, the present inventors concluded that this
bacterium does not belong to known genera but belongs to a novel
genus based on the 16S rDNA sequence homology, and designated it as
Fucanobacter lyticus SN-1009. As used herein, Alteromonas sp.
SN-1009 is the same as Fucanobacter lyticus SN-1009.
This bacterial strain is designated as Alteromonas sp. SN-1009 and
deposited on Feb. 13, 1996 (date of original deposit) at
International Patent Organism Depositary, National Institute of
Advanced Industrial Science and Technology, AIST Tsukuba Central 6,
1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566, Japan.
under accession number FERM BP-5747 (transmitted to international
depositary authority under Budapest Treaty on Nov. 15, 1996). The
16S rDNA sequence of this bacterial strain is shown in SEQ ID NO:
1.
Thus, the sulfated fucan-digesting enzyme used according to the
present invention can be produced by culturing a bacterium that is
determined to belong to the same genus as Fucanobacter lyticus
SN-1009 based on the 16S rDNA sequence. According to the present
invention, Alteromonas sp. SN-1009, a spontaneous or artificial
mutant of Alteromonas sp. SN-1009 and microorganisms belonging to
genus Alteromonas and genus Fucanobacter capable of producing the
sulfated fucan-digesting enzyme used according to the present
invention can be utilized.
The sulfated fucan-digesting enzyme to be used according to the
present invention can be obtained from the above-mentioned
microorganism according to the method as described in Example
1.
The sulfated fucan oligosaccharide of the present invention can be
prepared by allowing a sulfated fucan-digesting enzyme to act on a
sulfated fucan-containing material. For example, a partially
purified preparation of sulfated fucan, a sulfated
fucose-containing polysaccharide fraction derived from a brown
alga, a product obtained by extracting a brown alga with an aqueous
solvent, or a brown alga itself can be preferably used as a
sulfated fucan-containing material.
For preparing the sulfated fucan oligosaccharide of the present
invention, a sulfated fucan or a sulfated fucan-containing material
may be dissolved according to a conventional method. The sulfated
fucan of the present invention or the sulfated fucan-containing
material may be dissolved in the solution at the maximal
concentration. However, the concentration is usually selected
taking its operationality and the amount of the sulfated
fucan-digesting enzyme according to the present invention used in a
reaction into consideration. The solvent for the sulfated fucan may
be selected from water, buffers and the like depending on the
objects. Usually, the pH of the solution is nearly neutral. The
enzymatic reaction is usually carried out at about 30.degree. C.
The molecular weight of the sulfated fucan oligosaccharide can be
controlled by adjusting the ratio or amount of the sulfated
fucan-digesting enzyme according to the present invention used in
the reaction, the composition of the reaction mixture, the reaction
time and the like. The sulfated fucan oligosaccharide of the
present invention having more homogeneous molecular weight
distribution or more homogeneous charge density distribution can be
prepared by subjecting the sulfated fucan oligosaccharide of the
present invention obtained as described above to molecular weight
fractionation or fractionation using an anion exchange column. A
conventional means for molecular weight fractionation can be
applied. For example, gel filtration or molecular weight
fractionation membrane may be used. Optionally, the smaller
molecules may be further purified using ion-exchange resin
treatment, active carbon treatment or the like, or they may be
desalted, sterilized or lyophilized. Thus, the sulfated fucan
oligosaccharide of the present invention having a structure so
homogeneous that one can determine the structure by NMR analysis as
described below can be obtained.
Although it is not intended to limit the present invention, for
example, a sulfated fucan oligosaccharide having a chemical
structure selected from the group consisting of general formulas
(II), (III), (XIII), (XIV), (XV) and (XVI): ##STR6##
wherein R is H or SO.sub.3 H and at least one of Rs is SO.sub.3 H
in all formulas above,
or a salt thereof exemplifies the sulfated fucan oligosaccharide of
the present invention.
The sulfated fucan oligosaccharides of the present invention have
sulfate groups within the molecules, which groups react with
various bases to form salts. The sulfated fucan oligosaccharide of
the present invention is stable when it is in a form of salt. It is
usually provided in a form of sodium and/or potassium and/or
calcium salt. The sulfated fucan oligosaccharide of the present
invention in a free form can be derived from a salt thereof by
utilizing cation-exchange resin such as Dowex 50W. Optionally, it
can be subjected to conventional salt-exchange to convert it into
any one of various desirable salts.
Pharmaceutically acceptable salts can be used as the salts of the
sulfated fucan oligosaccharide of the present invention. Examples
of the salts include salts with alkaline metals such as sodium and
potassium and alkaline earth metals such as calcium, magnesium and
zinc as well as ammonium salts.
Additionally, the sulfated fucan oligosaccharide of the present
invention can be used as a reagent for glycotechnology. For
example, a substance which is very useful as a reagent for
glycotechnology (e.g., which can be used as a fluorescence-labeled
standard for the sulfated fucan oligosaccharide) can be provided by
subjecting the oligosaccharide to 2-aminopyridine (PA)-labeling
according to the method as described in JP-B 5-65108 to prepare a
PA-labeled oligosaccharide.
EXAMPLES
The following examples further illustrate the present invention in
detail but are not to be construed to limit the scope thereof.
In the following examples, the molecular weights of the
oligosaccharides of sulfated fucan are their average molecular
weights that were calculated from the results of mass analyses of
them.
Referential Example 1
Preparation of Fucoidan from Kjellmaniella crassifolia
2 kg of dried cultured Kjellmaniella crassifolia was disrupted
using a cutter mill (Masuko Sangyo) equipped with a screen having a
pore diameter of 1 mm and suspended in 20 L of 80% ethanol. The
suspension was stirred at 25.degree. C. for 3 hours and filtered
through a filter paper. The residue was suspended in 40 L of 30 mM
phosphate buffer (pH 6.5) containing 100 mM sodium chloride. The
suspension was treated at 95.degree. C. for 2 hours and filtered
through a stainless steel screen having a pore diameter of 106
.mu.m. 200 g of active carbon, 4.5 L of ethanol and 12,000 U of
alginate lyase K (Nagase Biochemicals) were added to the filtrate.
The mixture was stirred at 25.degree. C. for 20 hours, and then
centrifuged. The supernatant was concentrated to 4 L using an
ultrafiltration device equipped with hollow fibers with exclusion
molecular weight of 100,000, centrifuged to remove insoluble
substances and allowed to stand at 5.degree. C. for 24 hours. The
formed precipitate was removed by centrifugation. The supernatant
was subjected to solvent exchange for 100 mM sodium chloride using
an ultrafiltration device. The solution was cooled to 4.degree. C.
or below, and the pH was adjusted to 2.0 with hydrochloric acid.
The formed precipitate was removed by centrifugation. The pH of the
supernatant was adjusted to 8.0 with sodium hydroxide. The
supernatant was concentrated to 4 L. The concentrate was subjected
to solvent exchange for 20 mM sodium chloride using an
ultrafiltration device. Insoluble substances in the solution were
removed by centrifugation. The supernatant was then lyophilized to
obtain 76 g of a dried fucoidan preparation from Kjellmaniella
crassifolia.
cl Referential Example 2
Preparation of Sulfated Fucan Fraction
7 g of the dried fucoidan preparation as described in Referential
Example 1 was dissolved in 700 ml of 20 mM imidazole-hydrochloride
buffer (pH 8.0) containing 50 mM sodium chloride and 10% ethanol
and centrifuged to remove insoluble substances. The supernatant was
loaded onto a DEAE-Cellulofine A-800 column (11.4.times.48 cm)
equilibrated with the same buffer. After washing with the same
buffer, elution was then carried out with a gradient of 50 mM to
1.95 M sodium chloride. Each fraction contained 250 ml of the
eluate. The total sugar content and the uronic acid content of each
fraction were measured according to the phenol-sulfuric acid method
and the carbazole-sulfuric acid method, respectively. The fractions
43-49, 50-55 and 56-67 were combined, desalted by electrodialysis
and lyophilized. 340 mg, 870 mg and 2.64 g of dried products were
obtained from the fractions 43-49, 50-55 and 56-67, respectively.
The fraction obtained from the fractions 56-67 was used as a
sulfated fucan fraction.
Referential Example 3
Method for Measuring Activity of Sulfated Fucan-digesting
Enzyme
12 .mu.l of a 2.5% solution of the sulfated fucan fraction, 60
.mu.l of 50 mM imidazole-hydrochloride buffer (pH 7.5), 9 .mu.l of
4 M sodium chloride, 6 .mu.l of 1 M calcium chloride, 21 .mu.l of
water and 12 .mu.l of a solution of the sulfated fucan-digesting
enzyme according to the present invention were mixed together.
After reacting at 30.degree. C. for 3 hours, the reaction mixture
was treated at 100.degree. C. for 10 minutes. After centrifugation,
100 .mu.l of the supernatant was analyzed using HPLC to determine
the degree of conversion into smaller molecules. As controls, a
reaction mixture obtained by a reaction in which the buffer used
for dissolving the sulfated fucan-digesting enzyme solution was
used in place of the digesting enzyme solution and a reaction
mixture obtained by a reaction in which water was used in place of
the sulfated fucan fraction were similarly analyzed using HPLC.
One unit of an activity of sulfated fucan-digesting enzyme is
defined as an amount of an enzyme that cleaves fucosyl bonds in 1
.mu.mol of a sulfated fucan in 1 minute in the above-mentioned
reaction system. The activity of sulfated fucan-digesting enzyme
was calculated according to the following equation:
12.times.1000.times.2.5/100: sulfated fucan fraction added (.mu.g);
MG: the average molecular weight of the sulfated fucan as a
substrate; M: the average molecular weight of the reaction product;
(MG/M)-1: the number of sites cleaved by the enzyme in one sulfated
fucan molecule; 180: the reaction time (minutes); and 0.012: the
volume of the enzyme solution (ml).
The HPLC was carried out as follows:
Instrument: L-6200 (Hitachi);
Column: OHpak SB-806HQ (8.times.300 mm; Showa Denko);
Eluent: 50 mM sodium chloride containing 5 mM sodium azide;
Detection: differential refractive index detector (Shodex RI-71,
Showa Denko);
Flow rate: 1 ml/minute; and
Column temperature: 25.degree. C.
The following procedure was carried out in order to determine the
average molecular weight of the reaction product. Commercially
available pullulan (STANDARD P-82, Showa Denko) of which the
molecular weight was known was analyzed under the same conditions
as those for the above-mentioned HPLC analysis. The relationship
between the molecular weight of pullulan and retention time was
expressed as a curve, which was used as a standard curve for
determining the molecular weight of the reaction product. The
amount of protein was determined by measuring the absorbance of the
enzyme solution at 280 nm. The calculation was carried out assuming
the absorbance of a solution containing a protein at a
concentration of 1 mg/ml as 1.0.
Example 1
Preparation of Sulfated Fucan-digesting Enzyme
Fucanobacter lyticus SN-1009 was inoculated into 4 ml of a medium
consisting of artificial seawater (Jamarine Laboratory) (pH 8.2)
containing 0.2% fucoidan prepared as described in Referential
Example 1 and 0.3% peptone which had been autoclaved at 120.degree.
C. for 20 minutes, and cultured at 25.degree. C. for 24 hours to
prepare a seed culture. The seed culture was inoculated into 600 ml
of a medium consisting of artificial seawater (pH 8.2) containing
0.25% glucose, 1% peptone, 0.05% yeast extract and antifoaming
agent (KM70, Shin-Etsu Chemical) in a 2-L Erlenmeyer flask which
had been autoclaved at 120.degree. C. for 20 minutes, and cultured
at 25.degree. C. for 20 hours. 20 L of a medium consisting of
artificial seawater (pH 8.2) containing 1% peptone, 0.02% yeast
extract and antifoaming agent (KM70, Shin-Etsu Chemical) in a 30-L
jar fermentor which had been autoclaved at 120.degree. C. for 20
minutes was mixed with the fucoidan prepared as described in
Referential Example 1 which had been treated at 100.degree. C. for
20 minutes. The culture was inoculated into the mixture and
cultured at 25.degree. C. for 28 hours. After cultivation, the
culture was centrifuged to collect cells and a culture
supernatant.
The culture supernatant was concentrated using an ultrafiltration
device equipped with hollow fibers with exclusion molecular weight
of 10,000. The concentrate was subjected to solvent exchange for 20
mM Tris-hydrochloride buffer (pH 8.2) containing 10 mM calcium
chloride and 150 mM sodium chloride and centrifuged to obtain a
supernatant.
The supernatant was loaded onto a 2-L DEAE-Cellulofine A-800 column
equilibrated with the same buffer. After washing with the same
buffer, elution was then carried out with a gradient of 150 mM to
400 mM sodium chloride such that each fraction contained 63 ml of
the eluate to collect an active fraction.
The active fraction was concentrated using an ultrafiltration
device equipped with hollow fibers with exclusion molecular weight
of 10,000. The concentrate was subjected to solvent exchange for 20
mM Tris-hydrochloride buffer (pH 8.2) containing 10 mM calcium
chloride and 100 mM sodium chloride. The enzyme solution was loaded
onto a 200-ml DEAE-Cellulofine A-800 column equilibrated with the
same buffer. After washing with the same buffer, elution was then
carried out with a gradient of 100 mM to 300 mM sodium chloride
such that each fraction contained 19 ml of the eluate to collect an
active fraction.
The active fraction was concentrated using an ultrafiltration
device equipped with an ultrafiltration membrane with exclusion
molecular weight of 10,000. Sodium chloride at a final
concentration of 4 M was added thereto. The solution was loaded
onto a Phenyl-Sepharose CL-4B column equilibrated with 20 mM
Tris-hydrochloride buffer (pH 8.0) containing 100 mM calcium
chloride and 4 M sodium chloride. After washing with the same
buffer, elution was then carried out with a gradient of 4 M to 1 M
sodium chloride such that each fraction contained 9.4 ml of the
eluate. Thus, a purified preparation of sulfated fucan-digesting
enzyme was obtained.
Example 2
Preparation of Sulfated Fucan Oligosaccharide Using Sulfated
Fucan-digesting Enzyme, and Purification and Structural Analysis
Thereof
(1) Preparation
200 g of dried cultured Kjellmaniella crassifolia was soaked in 10
L of 18 mM imidazole-hydrochloride buffer (pH 7.0) containing 45 mM
calcium chloride, 500 mM sodium chloride and 9% ethanol. 30 U of
the sulfated fucan-digesting enzyme was added thereto. The mixture
was stirred at room temperature for 2 days and filtered through a
filter paper. A small molecule fraction recovered from the filtrate
using an ultrafiltration device equipped with hollow fibers with
exclusion molecular weight of 100,000 was designated as a sulfated
fucan oligosaccharide fraction 1.
(2) Purification
The sulfated fucan oligosaccharide fraction 1 obtained in Example
2-(1) was desalted using a desalting apparatus (Micro Acilyzer G3,
Asahi Kasei) and concentrated using a rotary evaporator. Imidazole
and sodium chloride were added to the sulfated fucan
oligosaccharide solution 1 at final concentrations of 10 mM and 300
mM, respectively. The resulting mixture was loaded onto a 1-L
DEAE-Cellulofine A-800 column equilibrated with 10 mM
imidazole-hydrochloride buffer (pH 6.0) containing 300 mM sodium
chloride. After adequately washing with the same buffer, elution
was then carried out with a gradient of 300 mM to 1200 mM sodium
chloride. The total sugar content and the total uronic acid content
of each of the eluted fractions were measured according to the
phenol-sulfuric acid method and the carbazole-sulfuric acid method,
respectively. As a result, the eluted fractions formed at least
four distinct peaks. The fractions in the respective peaks were
subjected to mass spectrometric analyses. Determination of the
saccharide compositions for the respective peaks showed that they
contained only fucose but did not contain uronic acid. Compositions
of the oligosaccharides having the respective masses estimated
based on the saccharide compositions are shown in Table 1.
TABLE 1 Composition of oligosaccharide Molecular Sulfate Peak no.
weight Fucose group Sodium 1-(1) 1914 6 10 10 1812 6 9 9 1564 5 8 8
1-(2) 2264 7 12 12 2162 7 11 11 1914 6 10 10 2016 6 11 11 1-(3)
2366 7 13 13 2264 7 12 12 2016 6 11 11 1914 6 10 10 2162 7 11 11
1-(4) 3460 11 18 18 3358 11 17 17 3110 10 16 16
the fractions constituting each peak were combined, concentrated
using an evaporator and purified as follows:
Column: YMC Pack Polyamine II (20.times.250 mm, YMC);
Flow rate: 8 ml/minute.;
Column temperature: 30.degree. C.;
Equilibration solution: 0.5 M sodium dihydrogenphosphate containing
10% acetonitrile;
Elution solution: gradient from 0.5 M sodium dihydrogenphosphate
containing 10% acetonitrile to 1.5 M sodium dihydrogenphosphate
containing 10% acetonitrile; as for peak number 1-(4), the gradient
for the elution was from 787.5 mM sodium dihydrogenphosphate
containing 10% acetonitrile to 1462.5 mM sodium dihydrogenphosphate
containing 10% acetonitrile;
Fractionation: 4 ml/fraction; and
Detection: the phenol-sulfuric acid method.
Fractions obtained by the column chromatography were subjected to
mass spectrometric analyses. As a result, substances having masses
of 1914, 2264, 2366 and 3460 were observed as main peaks for the
peak nos. 1-(1), 1-(2), 1-(3) and 1-(4), respectively. Fractions
constituting each peak were combined, loaded onto a Cellulofine
GCL-25 column equilibrated with 10% ethanol and eluted using 10%
ethanol for desalting. Thus, the sulfated fucan oligosaccharides
1-(1) to 1-(4) of the present invention were obtained.
(3) Structural analysis
The sulfated fucan oligosaccharides 1-(1) to 1-(4) of the present
invention obtained in Example 2-(2) were subjected to analyses of
saccharides at the reducing ends and saccharide compositions
according to a fluorescence labeling method using 2-aminopyridine.
As a result, the saccharide at the reducing end of and the sole
saccharide constituting each of the oligosaccharides 1-(1) to 1-(4)
were determined to be fucose. Next, determination of the sulfuric
acid content (measured according to the turbidinetric method using
barium chloride) and the uronic acid content (measured according to
the carbazole-sulfuric acid method), and NMR analysis using a
nuclear magnetic resonance apparatus JNM .alpha.-500 (Nippon
Denshi) were carried out. Samples to be analyzed were subjected to
structural analyses after exchange for heavy water according to a
conventional method. Bonds of constituting saccharides were
analyzed using the HMBC method, a method for .sup.1 H-detection of
heteronuclei. The DQF-COSY method and the HOHAHA method were used
for identification in .sup.1 H-NMR. The HSQC method was used for
identification in .sup.13 C-NMR.
Physical properties of the oligosaccharides 1-(1) to 1-(4) are
shown below.
(a) Physical properties of the oligosaccharide 1-(1)
The results for mass spectrometric analysis and identification in
NMR analysis are shown below. The .sup.1 H-NMR spectrum, .sup.13
C-NMR spectrum and mass spectrum of the sulfated fucan
oligosaccharide 1-(1) of the present invention are illustrated in
FIGS. 1, 2 and 3, respectively. In FIGS. 1 and 2, the vertical axes
represent the signal intensity and the horizontal axes represent
the chemical shift value (ppm). In FIG. 3, the vertical axis
represents the relative intensity and the horizontal axis
represents the m/z value. Molecular weight: 1914 MS m/z 455.0
[M-4Na.sup.+ ].sup.4-, 614.8 [M-3Na.sup.+ ].sup.3-, 933.8
[M-2Na.sup.+ ].sup.2-
Results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Tables 2 and 3.
TABLE 2 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 91.3
5.41, d, 3.4 F1-2 75.9 4.39, dd, 3.4, 10.1 F1-3 75.9 4.14, dd, 2.4,
10.1 F1-4 81.1 4.81, d, 2.4 F1-5 67.6 4.20, q, 6.4 F1-6 16.8 1.16,
d, 6.4 F2-1 99.7 5.25, d, 1.8 F2-2 75.2 4.43, m F2-3 72.5 4.43, m
F2-4 78.8 4.75, br-s F2-5 68.3 4.27, q, 6.7 F2-6 17.0 1.18, d, 6.7
F3-1 91.0 5.22, br-s F3-2 75.8 4.10, d, 5.2 F3-3 74.7 4.27, dd,
5.2, 2.5 F3-4 74.0 4.73, dd, 2.5, 5.5 F3-5 71.9 4.32, m, 5.5, 6.1
F3-6 14.2 1.45, d, 6.1
TABLE 3 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F4-1 98.5
5.32, d, 3.1 F4-2 74.0 4.45, m F4-3 71.0 4.27, m F4-4 78.9 4.76,
br-s F4-5 68.3 4.29, q, 6.7 F4-6 17.0 1.18, d, 6.7 F5-1 96.1 5.29,
d, 3.4 F5-2 75.9 4.35, dd, 3.4, 10.7 F5-3 67.4 4.27, dd, 2.4, 10.7
F5-4 82.3 4.60, d, 2.4 F5-5 67.4 4.38, q, 6.4 F5-6 17.0 1.16, d,
6.4 F6-1 100.5 5.18, d, 4.0 F6-2 66.7 3.84, dd, 4.0, 10.4 F6-3 78.8
4.45, dd, 3.1, 10.4 F6-4 71.1 4.07, d, 3.1 F6-5 67.9 4.06, q, 6.4
F6-6 16.5 1.13, d, 6.4 Saccharide composition: L-fucose (6
molecules) Sulfate group: 10 molecules Sodium: 10 molecules
The numbers for peak identification in .sup.1 H-NMR and .sup.13
C-NMR are as indicated in formula (IV) below: ##STR7##
(b) Physical properties of the oligosaccharide 1-(2)
The results for mass spectrometric analysis and identification in
NMR analysis are shown below. The .sup.1 H-NMR spectrum, .sup.13
C-NMR spectrum and mass spectrum of the sulfated fucan
oligosaccharide 1-(2) of the present invention are illustrated in
FIGS. 4, 5 and 6, respectively. In FIGS. 4 and 5, the vertical axes
represent the signal intensity and the horizontal axes represent
the chemical shift value (ppm). In FIG. 6, the vertical axis
represents the relative intensity and the horizontal axis
represents the m/z value. Molecular weight: 2264 MS m/z 354.2
[M-6Na.sup.+ ].sup.6-, 429.8 [M-5Na.sup.+ ].sup.5-, 543.0
[M-4Na.sup.+ ].sup.4-, 731.6 [M-3Na.sup.+ ].sup.3-
Results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Tables 4 and 5.
TABLE 4 Chemical shift value (ppm) .sup.13 C-NMR .sup.1 H-NMR F1-1
90.3 5.41, d, 3.4 F1-2 75.7 4.38, dd, 3.4, 9.8 F1-3 74.9 4.22, m
F1-4 80.2 4.80, br-s F1-5 67.5 4.23, q, 6.7 F1-6 16.0 1.19, d, 6.7
F2-1 99.3 5.31, d, 3.4 F2-2 75.2 4.40, m F2-3 76.9 4.29, m F2-4
81.5 4.83, d, 2.8 F2-5 68.5 4.22, q, 6.4 F2-6 16.4 1.16, d, 6.4
F3-1 99.7 5.29, br-s F3-2 74.0 4.41, m F3-3 71.1 4.41, m F3-4 78.2
4.75, m F3-5 67.4 4.34, q, 6.7 F3-6 16.5 1.22, d, 6.7 F4-1 89.6
5.26, br-s F4-2 74.4 4.07, m F4-3 74.4 4.27, m F4-4 73.3 4.73, dd,
2.8, 5.8 F4-5 71.2 4.36, m, 5.8, 6.4 F4-6 13.5 1.44, d, 6.4
TABLE 5 Chemical shift value (ppm) .sup.13 C-NMR .sup.1 H-NMR F5-1
98.3 5.31, d, 3.4 F5-2 73.4 4.45, dd, 3.4, 10.1 F5-3 70.4 4.26, m
F5-4 78.2 4.76, br-s F5-5 67.6 4.33, q, 6.4 F5-6 16.4 1.23, d, 6.4
F6-1 95.2 5.29, d, 3.7 F6-2 75.2 4.36, m F6-3 66.8 4.27, m F6-4
81.7 4.60, d, 2.8 F6-5 66.8 4.38, q, 6.7 F6-6 16.4 1.16, d, 6.7
F7-1 100.1 5.17, d, 4.3 F7-2 66.1 3.86, dd, 4.3, 10.4 F7-3 78.3
4.45, dd, 3.7, 10.4 F7-4 70.5 4.07, d, 3.7 F7-5 67.4 4.05, q, 6.4
F7-6 16.0 1.13, d, 6.4 Saccharide composition: L-fucose (7
molecules) Sulfate group: 12 molecules Sodium: 12 molecules
The numbers for peak identification in .sup.1 H-NMR and .sup.13
C-NMR are as indicated in formula (V) below: ##STR8##
An activity of inducing HGF production as determined according to
the method described in Example 1-(2) in WO 00/62785 was observed
for the sulfated saccharide of formula (V).
(c) Physical properties of the oligosaccharide 1-(3)
The results for mass spectrometric analysis are shown below. The
.sup.1 H-NMR spectrum, .sup.13 C-NMR spectrum and mass spectrum of
the sulfated fucan oligosaccharide 1-(3) of the present invention
are illustrated in FIGS. 7, 8 and 9. In FIGS. 7 and 8, the vertical
axes represent the signal intensity and the horizontal axes
represent the chemical shift value (ppm) In FIG. 9, the vertical
axis represents the relative intensity and the horizontal axis
represents the m/z value. Molecular weight: 2366 MS m/z 371.4
[M-6Na.sup.+ ].sup.6-, 450.3 [M-5Na.sup.+ ].sup.5-, 568.6
[M-4Na.sup.+ ].sup.4-, 765.7 [M-3Na.sup.+ ].sup.3-
The results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Tables 6 and 7.
TABLE 6 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 89.6
5.42, d, 2.5 F1-2 75.3 4.40, m F1-3 73.9 4.28, m F1-4 78.6 4.78, m
F1-5 67.5 4.24, m F1-6 15.9 1.24, d, 6.5 F2-1 98.3 5.34, d, 3.5
F2-2 74.8 4.43, m F2-3 76.0 4.29, m F2-4 80.8 4.83, m F2-5 68.1
4.20, m F2-6 15.9 1.18, d, 7.0 F3-1 99.1 5.29, m F3-2 73.8 4.45, m
F3-3 70.8 4.45, m F3-4 77.5 4.73, m F3-5 66.8 4.31, m F3-6 15.9
1.25, d, 6.5
TABLE 7 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F4-1 89.2
5.27, br-s F4-2 74.1 4.09, d, 9.5 F4-3 74.3 4.26, m F4-4 72.8 4.74,
m F4-5 70.8 4.36, m F4-6 13.3 1.46, d, 7.0 F5-1 97.3 5.31, d, 3.5
F5-2 73.1 4.47, dd, 10.0, 3.5 F5-3 69.8 4.27, m F5-4 77.5 4.75, m
F5-5 67.0 4.34, m F5-6 15.9 1.20, d, 6.0 F6-1 94.6 5.29, m F6-2
74.9 4.38, m F6-3 66.6 4.29, m F6-4 81.3 4.60, m F6-5 66.4 4.38, m
F6-6 15.4 1.17, d, 6.5 F7-1 99.3 5.22, d, 4.0 F7-2 65.9 3.91, dd,
11.0, 4.0 F7-3 75.3 4.52, dd, 11.0, 3.0 F7-4 79.0 4.80, d, 3.0 F7-5
66.6 4.16, m F7-6 15.9 1.20, d, 6.0 Saccharide composition:
L-fucose (7 molecules) Sulfate group: 13 molecules Sodium: 13
molecules
The numbers for signal assignment in .sup.1 H-NMR and .sup.13 C-NMR
are as indicated in formula (VI) below: ##STR9##
(d) Physical properties of the oligosaccharide 1-(4)
The results for mass spectrometric analysis are shown below. The
.sup.1 H-NMR spectrum, .sup.13 C-NMR spectrum and mass spectrum of
the sulfated fucan oligosaccharide 1-(4) of the present invention
are illustrated in FIGS. 10, 11 and 12. In FIGS. 10 and 11, the
vertical axes represent the signal intensity and the horizontal
axes represent the chemical shift value (ppm). In FIG. 12, the
vertical axis represents the relative intensity and the horizontal
axis represents the m/z value. Molecular weight: 3460 MS m/z 409.6
[M-8Na.sup.+ ].sup.8-, 471.4 [M-7Na.sup.+ ].sup.7-, 553.8
[M-6Na.sup.+ ].sup.6-, 669.3 [M-5Na.sup.+ ].sup.5-, 842.3
[M-4Na.sup.+ ].sup.4-, 1130.8 [M-3Na.sup.+ ].sup.3-
The results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Tables 8 to 10.
TABLE 8 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 90.3
5.39, d, 3.3 F1-2 75.5 4.37, m F1-3 76.0 4.21, m F1-4 79.8 4.77, m
F1-5 67.5 4.19, m F1-6 16.5 1.15, m F2-1 99.8 5.28, m F2-2 73.6
4.41, m F2-3 73.6 4.41, m F2-4 78.1 4.74, m F2-5 68.7 4.25, m F2-6
16.8 1.20, m F3-1 98.7 5.31, m F3-2 74.8 4.46, m F3-3 73.6 4.35, m
F3-4 78.1 4.74, m F3-5 67.7 4.25, m F3-6 16.8 1.20, m F4-1 90.2
5.29, m F4-2 74.8 4.04, m F4-3 74.9 4.27, m F4-4 74.3 4.74, m F4-5
68.1 4.27, q, 6.5 F4-6 14.0 1.39, d, 6.5
TABLE 9 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F5-1 99.2
5.34, d, 3.5 F5-2 74.3 4.42, m F5-3 74.3 4.21, m F5-4 81.5 4.81, m
F5-5 68.7 4.20, m F5-6 16.6 1.18, m F6-1 98.7 5.31, m F6-2 75.3
4.46, m F6-3 71.1 4.35, m F6-4 78.1 4.74, m F6-5 67.7 4.25, m F6-6
16.7 1.19, m F7-1 90.2 5.29, m F7-2 74.8 4.04, m F7-3 74.9 4.27, m
F7-4 74.3 4.74, m F7-5 67.9 4.27, q, 7.0 F7-6 14.0 1.43, d, 7.0
TABLE 10 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F8-1 96.0
5.30, m F8-2 75.3 4.46, m F8-3 71.1 4.35, m F8-4 78.1 4.81, m F8-5
67.1 4.43, m F8-6 16.7 1.19, m F9-1 96.0 5.29, m F9-2 75.5 4.33, m
F9-3 67.1 4.27, m F9-4 82.0 4.58, d, 2.8 F9-5 67.1 4.34, q, 6.5
F9-6 16.5 1.15, d, 6.5 F10-1 99.8 5.19, d, 4.2 F10-2 66.3 3.85, dd,
4.2, 10.4 F10-3 78.5 4.43, m F10-4 70.8 4.05, br-s F10-5 67.5 4.03,
q, 6.6 F10-6 16.1 1.12, d, 6.6 F11-1 99.8 5.21, d, 4.2 F11-2 66.3
3.85, dd, 4.2, 10.4 F11-3 78.5 4.44, m F11-4 70.8 4.06, br-s F11-5
67.5 4.08, q, 6.6 F11-6 16.1 1.12, d, 6.6 Saccharide composition:
L-fucose (11 molecules) Sulfate group: 18 molecules Sodium: 18
molecules
The numbers for signal assignment in .sup.1 H-NMR and .sup.13 C-NMR
are as indicated in formula (VII) below: ##STR10##
Example 3
Preparation of Sulfated Fucan Oligosaccharide using Sulfated
Fucan-Digesting Enzyme, and Purification and Structural Analysis
Thereof
(1) Preparation
Chips were prepared from dried cultured Kjellmaniella crassifolia
using a cutter mill (Masuko Sangyo) equipped with a screen having a
pore diameter of 1 mm. 250 g of the Kjellmaniella crassifolia chips
were suspended in 5 L of 80% ethanol. The suspension was stirred at
room temperature for 2 hours and filtered. The washing in 80%
ethanol was repeated four times to obtain washed Kjellmaniella
crassifolia chips. 250 g of the washed chips were suspended in 5 L
of 17 mM imidazole-hydrochloride buffer (pH 7.5) containing 125 mM
calcium chloride, 250 mM sodium chloride and 10% ethanol. The
suspension was stirred at room temperature for 24 hours, filtered
and centrifuged to obtain a Kjellmaniella crassifolia fucoidan
solution. 10 U of the sulfated fucan-digesting enzyme was added to
1 L of the extract. The resulting mixture was stirred at room
temperature for three days, and then filtered through a filter
paper. A supernatant was obtained by centrifuging the filtrate. A
small molecule fraction recovered from the supernatant using an
ultrafiltration device equipped with hollow fibers with exclusion
molecular weight of 10,000 was designated as a sulfated fucan
oligosaccharide fraction 2.
(2) Purification
The sulfated fucan oligosaccharide fraction 2 obtained in Example
3-(1) was loaded onto a 1-L DEAE-Cellulofine A-800 column
equilibrated with 10 mM imidazole-hydrochloride buffer (pH 6.5)
containing 100 mM sodium chloride. After adequately washing with
the same buffer, elution was then carried out with a gradient of
100 mM to 1 M sodium chloride. The total sugar content and the
total uronic acid content of each of the eluted fractions were
measured according to the phenol-sulfuric acid method and the
carbazole-sulfuric acid method, respectively. As a result, the
eluted fractions formed two peaks. The fractions in the respective
peaks were subjected to mass spectrometric analyses. Determination
of the saccharide compositions for the respective peaks showed that
they contained only fucose but did not contain uronic acid.
Compositions of the oligosaccharides having the respective masses
estimated based on the saccharide compositions are shown in Table
11.
TABLE 11 Composition of oligosaccharide Molecular Sulfate Peak no.
weight Fucose group Sodium 2-(1) 1914 6 10 10 2264 7 12 12 2016 6
11 11 2-(2) 3110 10 16 16 2760 9 14 14 3360 11 17 17 4308 14 22 22
3958 13 20 20
The fractions constituting each peak were pooled, concentrated
using an evaporator, and purified by the same method described in
Example 2-(2).
Fractions obtained by YMC Pack Polyamine II column chromatography
were subjected to mass spectrometric analyses, and substances
having masses of 1914 and 2016 from peak number 2-(1), and a
substance having a mass of 3110 from peak number 2-(2) were
obtained as their main peaks.
The fractions constituting each peak that contained substances
having masses of 1914, 2016, and 3110 were pooled, applied to a
Cellulofine GCL-25 column equilibrated with 10% ethanol and eluted
using 10% ethanol to desalt. Thus the sulfated fucan
oligosaccharides 2-(1)-1, 2-(1)-2, and 2-(2) of the present
invention were obtained.
(3) Structural Analyses
The sulfated fucan oligosaccharides 2-(1)-1, 2-(1)-2, and 2-(2) of
the present invention obtained in Example 3-(2) were subjected to
analyses of the reducing terminal sugar and component sugar
according to a fluorescence labeling method using 2-aminopyridine.
As a result, the reducing terminal and the component sugar of them
were only fucose. Next, their sulfate content (by the turbidimetric
method using barium chloride) and their uronic acid content (by the
carbazole-sulfuric acid method) were determined. NMR analyses of
them using a nuclear magnetic resonance apparatus JNM .alpha.-500
(Nippon Denshi) were also carried out. The linkages among the
constituting saccharides were analyzed using the HMBC method, a
method for .sup.1 H-detection of heteronuclei. The DQF-COSY method
and the HOHAHA method were used for the assignment of .sup.1 H-NMR
data, and the HSQC method was used for the assignment of .sup.13
C-NMR data.
Physical properties of the sulfated fucan oligosaccharides 2-(1)-1,
2-(1)-2, and 2-(2) of the present invention are shown below.
(a) Physical properties of the sulfated fucan oligosaccharide
2-(1)-1
The result of all the above analyses showed that the sulfated fucan
oligosaccharide 2-(1)-1 is the same substance as the sulfated fucan
oligosaccharide 1-(1) of the present invention.
(b) Physical properties of the sulfated fucan oligosaccharide
2-(1)-2
The results for the mass spectrometric analysis and assignment in
NMR analyses are shown below. The .sup.1 H-NMR spectrum, .sup.13
C-NMR spectrum, and the mass spectrum of this oligosaccharide are
shown in FIGS. 13, 14, and 15, respectively. In FIGS. 13 and 14,
the vertical axes represent the signal intensity and the horizontal
axes represent the chemical shift value (ppm). In FIG. 15, the
vertical axis represents the relative intensity of the signals and
the horizontal axis represents the m/z value. Molecular weight:
2016 MS m/z 313.1 [M-6Na.sup.+ ].sup.6-, 380.3 [M-5Na.sup.+
].sup.5-, 481.1 [M-4Na.sup.+ ].sup.4-, 649.1 [M-3Na.sup.+ ].sup.3-,
985.0 [M-2Na.sup.+ ].sup.2-
The results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Tables 12 and 13.
TABLE 12 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 90.7
5.43, d, 3.6 F1-2 75.3 4.41, dd, 3.6, 10.1 F1-3 75.7 4.15, dd,
10.1, 2.4 F1-4 80.6 4.83, d, 2.4 F1-5 67.0 4.22, q, 6.6 F1-6 16.5
1.18, d, 6.6 F2-1 99.2 5.27, d, 3.2 F2-2 74.5 4.45, m F2-3 71.9
4.46, m F2-4 78.1 4.77, br-s F2-5 67.7 4.30, q, 6.6 F2-6 16.5 1.20,
d, 6.6 F3-1 89.8 5.24, br-s F3-2 75.2 4.12, m F3-3 74.2 4.28, m
F3-4 73.3 4.76, br-s F3-5 71.4 4.36, q, 6.0 F3-6 13.6 1.48, d,
6.0
TABLE 13 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F4-1 98.2
5.33, d, 3.3 F4-2 73.3 4.47, m F4-3 71.0 4.28, m F4-4 78.1 4.78,
br-s F4-5 67.7 4.32, q, 6.6 F4-6 16.5 1.20, d, 6.6 F5-1 94.8 5.31,
d, 3.4 F5-2 75.3 4.38, m F5-3 66.9 4.28, m F5-4 81.7 4.62, d, 2.4
F5-5 66.8 4.40, q, 6.6 F5-6 16.5 1.18, d, 6.6 F6-1 100.1 5.22, d,
3.6 F6-2 66.3 3.89, dd, 3.6, 10.4 F6-3 75.7 4.52, dd, 10.4, 3.0
F6-4 79.2 4.82, d, 3.0 F6-5 67.2 4.18, q, 6.6 F6-6 16.3 1.20, d,
6.6 Component sugar: only L-fucose (6 molecules) Sulfate residues:
11 molecules Sodium: 11 molecules
The numbers for signal assignment in .sup.1 H-NMR and .sup.13 C-NMR
analyses are as indicated in formula (VIII) below: ##STR11##
Physical properties of the oligosaccharide 2-(2)
The results of the mass spectrometric analysis are shown below, and
the .sup.1 H-NMR spectrum, .sup.13 C-NMR spectrum, and the mass
spectrum of the sulfated fucan oligosaccharide 2-(2) of the present
invention are shown in FIGS. 16, 17, and 18, respectively. In FIGS.
16 and 17, the vertical axes represent the signal intensity and the
horizontal axes represent the chemical shift value (ppm). In FIG.
18, the vertical axis represents the relative intensity of the
signals and the horizontal axis represents the m/z value. Molecular
weight: 3111 MS m/z 365.8[M-8Na.sup.+ ].sup.8-, 421.4[M-7Na.sup.+
].sup.7-, 495.2[M-6Na.sup.+ ].sup.6-, 599.1[M-5Na.sup.+ ].sup.5-,
755.0[M-4Na.sup.+ ].sup.4-, 1013.7[M-3Na.sup.+ ].sup.3-
The results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Tables 14 to 16.
TABLE 14 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 90.8
5.44, d, 3.4 F1-2 75.7 4.44, dd, 3.4, 9.8 F1-3 75.9 4.19, dd, 9.8,
2.9 F1-4 80.3 4.84, d, 2.9 F1-5 67.7 4.24, m F1-6 16.6 1.22, m F2-1
99.4 5.30, d, 3.0 F2-2 74.9 4.46, m F2-3 72.5 4.44, m F2-4 78.4
4.78, br-s F2-5 68.2 4.29, q, 6.6 F2-6 17.1 1.25, d, 6.6 F3-1 91.2
5.29, br-s F3-2 74.9 4.11, m F3-3 75.0 4.28, m F3-4 74.5 4.78, br-s
F3-5 68.4 4.31, q, 6.8 F3-6 14.5 1.43, d, 6.8 F4-1 98.7 5.40, d,
3.5 F4-2 74.9 4.48, m F4-3 74.9 4.29, m F4-4 80.3 4.48, br-s F4-5
68.4 4.24, m F4-6 16.6 1.22, m
TABLE 15 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F5-1 98.0
5.35, d, 3.6 F5-2 73.8 4.51, m F5-3 71.2 4.39, m F5-4 78.4 4.78, m
F5-5 68.2 4.29, m F5-6 16.9 1.23, m F6-1 90.5 5.34, br-s F6-2 74.9
4.11, m F6-3 75.0 4.29, m F6-4 74.5 4.78, br-s F6-5 68.4 4.31, q,
7.0 F6-6 14.2 1.48, d, 7.0 F7-1 95.9 5.33, m F7-2 73.9 4.48, m F7-3
71.2 4.37, m F7-4 78.2 4.78, m F7-5 67.3 4.46, m F7-6 16.8 1.23,
m
TABLE 16 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F8-1 96.2
5.33, d, 3.3 F8-2 75.7 4.38, m F8-3 67.3 4.31, m F8-4 82.2 4.62, d,
3.1 F8-5 67.3 4.38, m F8-6 16.4 1.20, m F9-1 99.7 5.25, d, 4.4 F9-2
66.5 3.88, dd, 4.4, 10.2 F9-3 78.7 4.48, m F9-4 70.9 4.10, m F9-5
67.5 4.08, q, 6.6 F9-6 16.2 1.17, d, 6.6 F10-1 99.7 5.26, d, 4.4
F10-2 66.5 3.90, dd, 4.4, 10.2 F10-3 78.7 4.49, m F10-4 70.9 4.11,
m F10-5 67.5 4.12, q, 6.6 F10-6 16.3 1.17, d, 6.6 Saccharide
composition: L-fucose (10 molecules) Sulfate group: 16 molecules
Sodium: 16 molecules
The numbers for signal assignment in .sup.1 H-NMR and .sup.13 C-NMR
analyses are as indicated in formula (IX) below: ##STR12##
Example 4
The Preparation of Laminaria japonica Sulfated Fucan
Oligosaccharides using Sulfated Fucan-digesting Enzyme, Their
Purification, and Their Structural Analyses
(1) Preparation
Dried cultured Laminaria japonica was disrupted into chips using a
cutter mill (Masuko Sangyo) equipped with a screen having a pore
diameter of 1 mm. Five hundred grams of Laminaria japonica chips
were suspended in 4.5 L of 80% ethanol, stirred at room temperature
for 24 hours, and filtered. The residue was washed with 80% ethanol
for three times by the same method as described above, and the
washed Laminaria japonica chips were obtained. All the washed chips
were suspended in 10 L of 50 mM imidazole-HCl buffer (pH 8.0)
containing 50 mM calcium chloride, 200 mM sodium chloride, and 10%
ethanol, to the suspension was added 2 U of the sulfated
fucan-digesting enzyme, stirred at room temperature for 5 days,
filtered through filter paper, thus obtained filtrate was
centrifuged, and the supernatant was fractionated using an
ultrafiltration device equipped with hollow fibers with exclusion
molecular weight of 10,000, and the low molecular weight fraction
was recovered and designated as a Laminaria japonica sulfated fucan
oligosaccharide fraction 1.
(2) Purification
The conductivity of the solution of the Laminaria japonica sulfated
fucan oligosaccharide fraction 1 obtained in Example 4-(1) was
adjusted by adding 10% ethanol to the same conductivity of the
equilibration buffer described below, and it was applied to 500 ml
of DEAE-Cellulofine A-800 column equilibrated with 20 mM
imidazole-HCl buffer (pH 6.5) containing 200 mM sodium chloride and
10% ethanol, then the column was washed with the same buffer, and
eluted with a linear gradient of sodium chloride from 200 mM to 1.2
M. For the elution, 4.5 L of the buffer was used, and fractions of
50 ml were collected. The total sugar content and the total uronic
acid content of each of the eluted fractions were measured by the
phenol-sulfuric acid method and carbazole-sulfuric acid method,
respectively. As a result, the eluted fractions formed 3 peaks.
Fractions around the second peak (44-48) were pooled, concentrated
to 40 ml by evaporator, applied to Cellulofine GCL-25 column
(4.1.times.90 cm) equilibrated with 10% ethanol, and eluted with
10% ethanol. The total sugar of each of the eluted fractions was
determined by phenol-sulfuric acid method. The fractions in which
sugar was detected were pooled, concentrated to 8.4 ml by
evaporator, and purified by the condition described below.
Column: YMC Pack Polyamine II (20.times.250 mm, YMC)
Flow rate: 8 ml/minute
Column temperature: 30.degree. C.
Equilibration solution: 875 mM sodium dihydrogenphosphate
containing 10% acetonitrile
Elution: Gradient from 875 mM sodium dihydrogenphosphate containing
10% acetonitrile to 1.4 M sodium dihydrogenphosphate containing 10%
acetonitrile
Fractionation: 4 ml/fraction
Detection: by the phenol-sulfuric acid method
Fractions obtained by the column chromatography shown above (3
times, fraction number around 50-59) were pooled, dialyzed against
10% ethanol using a dialysis tube of which the exclusion molecular
weight is 3,500, concentrated to about 40 ml by evaporator, applied
to Cellulofine GCL-25 column equilibrated with 10% ethanol, and
eluted with 10% ethanol.
The total sugar of each of the eluted fractions was determined by
the phenol-sulfuric acid method. The fractions around the main peak
were pooled and purified again by Cellulofine GCL-25 as described
above. The fractions around the main peak of these eluted fractions
were pooled, concentrated by Speed Vac, and lyophilized. Thus
Laminaria japonica sulfated fucan oligosaccharide 1 was
obtained.
(3)Structural Analyses
By the structural analyses of the oligosaccharide as shown in
Example 2, the structure of Laminaria japonica sulfated fucan
oligosaccharide 1 was determined to be the same structure as the
sulfated fucan oligosaccharide 1-(3) shown in Example 2.
Example 5
Preparation, Purification, and Structural Analyses of Lessonia
nigrescence Sulfated Fucan Oligosaccharide
(1) Preparation
By the method shown in Referential Example 1, Lessonia nigrescens
fucoidan was prepared from dried chips of Lessonia nigrescens.
Namely, 10 g of Lessonia nigrescens fucoidan was dissolved in 2 L
of 50 mM imidazole-HCl buffer (pH 8.0) containing 50 mM calcium
chloride, 300 mM sodium chloride, and 10% ethanol. To the solution
was added 1 U of sulfated fucan-digesting enzyme, stirred at room
temperature for 40 hours, and recovered its low molecular weight
fraction using an ultrafiltration device equipped with hollow
fibers with exclusion molecular weight of 10,000, and thus obtained
fraction was designated as a Lessonia nigrescens sulfated fucan
oligosaccharide fraction 1.
(2) Purification
Lessonia nigrescens sulfated fucan oligosaccharide fraction 1
obtained in (1) was applied to 1000 ml of DEAE-Cellulofine A-800
column equilibrated with 10 mM imidazole-HCl buffer (pH 6.0)
containing 200 mM sodium chloride and 10% ethanol, then the column
was washed with the same buffer, and eluted with a linear gradient
of sodium chloride from 200 mM to 700 mM. For the elution, 5 L of
the buffer was used, and fractions of 56 ml were collected. The
total sugar content and the total uronic acid content of each of
the eluted fractions were measured by the phenol-sulfuric acid
method and carbazole-sulfuric acid method, respectively. As a
result, the eluted fractions formed 7 peaks. Fractions around the
each peak were pooled separately, concentrated to 40 ml by
evaporator (they are designated as Lessonia nigrescens sulfated
fucan oligosaccharides 1-(1) to 1-(7)), applied to Cellulofine
GCL-25 column (4.1.times.90 cm) equilibrated with 10% ethanol, and
eluted with 10% ethanol. The total sugar of each of the eluted
fractions was determined by phenol-sulfuric acid method. The
fractions in which sugar was detected were pooled, concentrated by
evaporator, and purified by the condition described below.
Column: YMC Pack Polyamine II (20.times.250 mm, YMC)
Flow rate: 8 ml/minute
Column temperature: 30.degree. C.
Equilibration solution: For Lessonia nigrescens sulfated fucan
oligosaccharides 1-(1), (2), and (3), 90 mM sodium
dihydrogenphosphate containing 10% acetonitrile. For Lessonia
nigrescens sulfated fucan oligosaccharide 1-(4) and (5), 630 mM
sodium dihydrogenphosphate containing 10% acetonitrile. For
Lessonia nigrescens sulfated fucan oligosaccharide 1-(6), 720 mM
sodium dihydrogenphosphate containing 10% acetonitrile. For
Lessonia nigrescens sulfated fucan oligosaccharide 1-(7), 900 mM
sodium dihydrogenphosphate containing 10% acetonitrile.
Elution: For Lessonia nigrescens sulfated fucan oligosaccharides
1-(1), (2), and (3), with the gradient from 90 mM sodium
dihydrogenphosphate containing 10% acetonitrile to 900 mM sodium
dihydrogenphosphate containing 10% acetonitrile. For Lessonia
nigrescens sulfated fucan oligosaccharides 1-(4) and (5), with the
gradient from 630 mM sodium dihydrogenphosphate containing 10%
acetonitrile to 1260 mM sodium dihydrogenphosphate containing 10%
acetonitrile. For Lessonia nigrescens sulfated fucan
oligosaccharide 1-(6), with the gradient from 720 mM sodium
dihydrogenphosphate containing 10% acetonitrile to 1440 mM sodium
dihydrogenphosphate containing 10% acetonitrile. For Lessonia
nigrescens sulfated fucan oligosaccharide 1-(7), with the gradient
from 900 mM sodium dihydrogenphosphate containing 10% acetonitrile
to 1620 mM sodium dihydrogenphosphate containing 10%
acetonitrile.
Fractionation: 4 ml/fraction
Detection: by the phenol-sulfuric acid method
Lessonia nigrescens sulfated fucan oligosaccharides from 1-(1) to
1-(7) were fractionated by the column chromatography shown above,
and the fractions in which sugar was detected were pooled,
concentrated to about 40 ml by evaporator, applied to Cellulofine
GCL-25 column equilibrated with 10% ethanol, and eluted with 10%
ethanol.
The total sugar of each of the eluted fractions was determined by
the phenol-sulfuric acid method. The fractions around the main peak
were pooled and purified again by Cellulofine GCL-25 as described
above. As for Lessonia nigrescens sulfated fucan oligosaccharide
1-(7), two main peaks were observed, therefore, the two peaks were
designated as Lessonia nigrescens sulfated fucan oligosaccharides
1-(7)-1 and 1-(7)-2.
The fractions around the main peak of each of these eluted
fractions were pooled, concentrated by Speed Vac, and lyophilized.
Thus Lessonia nigrescens sulfated fucan oligosaccharides from 1-(1)
to 1-(7)-2 were obtained.
(3) Structural Analyses
The structural analyses of these oligosaccharides were carried out
as described in Example 2.
Physical properties of the Lessonia nigrescens sulfated fucan
oligosaccharides 1-(1) to 1-(7)-2 of the present invention are
shown below.
(a) Physical Properties of Lessonia nigrescens Sulfated Fucan
Oligosaccharide 1-(1)
The results for the mass spectrometric analysis and assignment in
NMR analyses are shown below, and the .sup.1 H-NMR spectrum,
.sup.13 C-NMR spectrum, and the mass spectrum of this
oligosaccharide are shown in FIGS. 19, 20, and 21, respectively. In
FIGS. 19 and 20, the vertical axes represent the signal intensity
and the horizontal axes represent the chemical shift value (ppm).
In FIG. 21, the vertical axis represents the relative intensity of
the signals and the horizontal axis represents the m/z value.
Molecular weight; 718 MS m/z 216.5 [M-3Na.sup.+ ].sup.3-, 336.0
[M-2Na.sup.+ ].sup.2-, 695.0 [M-Na.sup.+ ].sup.-
The results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Table 17.
TABLE 17 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 90.8
5.41, d, 3.6 F1-2 75.1 4.38, dd, 3.6, 10.8 F1-3 75.1 4.11, dd,
10.8, 3.0 F1-4 81.0 4.84, d, 3.0 F1-5 66.7 4.22, q, 6.0 F1-6 16.2
1.16, d, 6.0 F2-1 99.2 5.21, d, 3.0 F2-2 76.4 4.29, m F2-3 67.8
4.29, m F2-4 81.5 4.55, d, 3.0 F2-5 67.8 4.31, q, 6.6 F2-6 16.3
1.16, d, 6.6 Component sugar: only L-fucose (2 molecules) Sulfate
residues: 4 molecules Sodium: 4 molecules
The numbers for signal assignment in .sup.1 H-NMR and .sup.13 C-NMR
analyses are as indicated in formula (X) below: ##STR13##
(b) Physical properties of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(2)
The results for the mass spectrometric analysis and assignment in
NMR analyses are shown below, and the .sup.1 H-NMR spectrum,
.sup.13 C-NMR spectrum, and the mass spectrum of the Lessonia
nigrescens sulfated fucan oligosaccharide 1-(2) are shown in FIGS.
22, 23, and 24, respectively. In FIGS. 22 and 23, the vertical axes
represent the signal intensity and the horizontal axes represent
the chemical shift value (ppm). In FIG. 24, the vertical axis
represents the relative intensity of the signals and the horizontal
axis represents the m/z value. Molecular weight; 966 MS m/z
459.9[M-2Na.sup.+ ].sup.2-, 942.9[M-Na.sup.+ ].sup.-
The results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Table 18.
TABLE 18 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 90.6
5.42, d, 3.6 F1-2 75.7 4.22, dd, 3.6, 10.4 F1-3 73.8 4.24, dd,
10.4, 2.4 F1-4 80.5 4.79, d, 2.4 F1-5 66.9 4.24, q, 6.4 F1-6 16.3
1.18, d, 6.4 F2-1 98.3 5.32, d, 2.0 F2-2 74.9 4.41, m F2-3 72.5
4.41, m F2-4 79.8 4.72, br-s F2-5 67.8 4.30, q, 6.5 F2-6 16.3 1.17,
d, 6.5 F3-1 97.7 5.03, d, 3.9 F3-2 69.0 3.66, dd, 3.9, 10.5 F3-3
69.3 3.93, dd, 10.5, 2.8 F3-4 81.4 4.51, d, 2.8 F3-5 66.9 4.46, q,
6.4 F3-6 16.3 1.19, d, 6.4 Component sugar: only L-fucose (3
molecules) Sulfate residues: 5 molecules Sodium: 5 molecules
The numbers for signal assignment in .sup.1 H-NMR and .sup.13 C-NMR
analyses are as indicated in formula (XI) below: ##STR14##
(c) Physical properties of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(3)
The results for the mass spectrometric analysis and assignment in
NMR analyses are shown below, and the .sup.1 H-NMR spectrum,
.sup.13 C-NMR spectrum, and the mass spectrum are shown in FIGS.
25, 26, and 27, respectively. In FIGS. 25 and 26, the vertical axes
represent the signal intensity and the horizontal axes represent
the chemical shift value (ppm). In FIG. 27, the vertical axis
represents the relative intensity of the signals and the horizontal
axis represents the m/z value. Molecular weight; 1068 MS m/z 332.9
[M-3Na.sup.+ ].sup.3-, 511.0 [M-2Na.sup.+ ].sup.2-, 1045.0
[M-Na.sup.+ ].sup.-
The results of .sup.1 H-NMR and .sup.13 C-NMR analyses are shown in
Table 19.
TABLE 19 Chemical shift value (ppm) .sup.1 H-NMR Chemical shift
value, multiplicity, .sup.13 C-NMR coupling constant F1-1 90.3
5.42, d, 3.4 F1-2 75.5 4.39, dd, 3.4, 10.2 F1-3 74.6 4.22, dd,
10.2, 2.9 F1-4 78.0 4.80, d, 2.9 F1-5 67.3 4.24, q, 6.6 F1-6 16.3
1.20, d, 6.6 F2-1 98.8 5.30, d, 3.3 F2-2 75.0 4.41, m F2-3 75.0
4.30, m F2-4 80.7 4.81, d, 2.6 F2-5 68.4 4.25, q, 6.7 F2-6 16.2
1.18, d, 6.7 F3-1 98.5 5.24, d, 3.2 F3-2 75.7 4.31, dd, 3.2, 10.4
F3-3 66.5 4.26, dd, 10.4, 3.0 F3-4 81.7 4.57, d, 3.0 F3-5 67.4
4.36, q, 6.6 F3-6 16.0 1.20, d, 6.6 Component sugar: only L-fucose
(3 molecules) Sulfate residues: 6 molecules Sodium: 6 molecules
The numbers for signal assignment in .sup.1 H-NMR and .sup.13 C-NMR
analyses are as indicated in formula (XII) below: ##STR15##
(d) Physical properties of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(4)
By the structural analyses of the oligosaccharide as shown in
Example 2, the structure of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(4) was determined to be the same structure as
the sulfated fucan oligosaccharide 1-(2) shown in Example 2.
(e) Physical properties of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(5)
By the structural analyses of the oligosaccharide as shown in
Example 2, the structure of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(5) was determined to be the same structure as
the sulfated fucan oligosaccharide 1-(3) shown in Example 2.
(f) Physical properties of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(6)
The results of the mass spectrometric analysis for Lessonia
nigrescens sulfated fucan oligosaccharides 1-(6) of the present
invention are shown below. Molecular weight; 3461 MS m/z 361.5
[M-9Na.sup.+ ].sup.9-, 409.62 [M-8Na.sup.+ ].sup.8-, 471.42
[M-7Na.sup.+ ].sup.7-, 553.81 [M-6Na.sup.+ ].sup.6-, 669.31
[M-5Na.sup.+ ].sup.5-, 842.32 [M-4Na.sup.+ ].sup.4-, 1130.83
[M-3Na.sup.+ ].sup.3- Component sugar: only L-fucose (11 molecules)
Sulfate residues: 18 molecules Sodium: 18 molecules
(g) Physical properties of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(7)-1
The results of the mass spectrometric analysis for Lessonia
nigrescens sulfated fucan oligosaccharide 1-(7)-1 of the present
invention are shown below. Molecular weight; 4659 MS m/z 442.82
[M-10Na.sup.+ ].sup.10-, 494.72 [M-9Na.sup.+ ].sup.9-, 559.32
[M-8Na.sup.+ ].sup.8-, 642.62 [M-7Na.sup.+ ].sup.7-, 753.52
[M-6Na.sup.+ ].sup.6-, 908.82 [M-5Na.sup.+ ].sup.5-, 1141.43
[M-4Na.sup.+ ].sup.4- Component sugar: only L-fucose (15 molecules)
Sulfate residues: 24 molecules Sodium: 24 molecules
(h) Physical properties of Lessonia nigrescens sulfated fucan
oligosaccharide 1-(7)-2
The results of the mass spectrometric analysis for Lessonia
nigrescens sulfated fucan oligosaccharide 1-(7)-2 of the present
invention are shown below. Molecular weight; 3564 MS m/z
373.12[M-9Na.sup.+ ].sup.9-, 422.52 [M-8Na.sup.+ ].sup.8-, 486.32
[M-7Na.sup.+ ].sup.7-, 571.01 [M-6Na.sup.+ ].sup.6-, 689.61
[M-5Na.sup.+ ].sup.5-, 868.02 [M-4Na.sup.+ ].sup.4-, 1164.93
[M-3Na.sup.+ ].sup.3- Component sugar: only L-fucose (11 molecules)
Sulfate residues: 19 molecules Sodium: 19 molecules
Example 6
Preparation of Sulfated Fucan Oligosaccharide Using Sulfated
Fucan-digesting Enzyme, and Purification and Structural Analysis
Thereof
(1) Preparation
10 g of the fucoidan from cultured Kjellmaniella crassifolia as
described in Referential Example 1 was dissolved in 4.8 L of 17 mM
imidazole-hydrochloride buffer (pH 7.5) containing 125 mM calcium
chloride, 250 mM sodium chloride and 10% ethanol. 10 U of the
sulfated fucan-digesting enzyme was added thereto. The resulting
mixture was stirred at room temperature for 72 hours to obtain a
Kjellmaniella crassifolia fucoidan oligosaccharide solution. A
small molecule fraction recovered from the solution using an
ultrafiltration device equipped with hollow fibers with exclusion
molecular weight of 10,000 was designated as a sulfated fucan
oligosaccharide fraction 3.
(2) Analysis
The sulfated fucan oligosaccharide fraction 3 obtained in (1) above
was loaded onto a 1-L DEAE-Cellulofine A-800 column equilibrated
with 10 mM imidazole-hydrochloride buffer (pH 6.5) containing 100
mM sodium chloride. After adequately washing with the same buffer,
elution was then carried out with a gradient of 100 mM to 1 M
sodium chloride. The total sugar content and the total uronic acid
content of each of the eluted fractions were measured according to
the phenol-sulfuric acid method and the carbazole-sulfuric acid
method, respectively. As a result, the eluted fractions formed two
peaks. The fractions in the respective peaks were subjected to mass
spectrometric analyses. Determination of the saccharide
compositions for the respective peaks showed that they contained
only fucose but did not contain uronic acid. Compositions of the
oligosaccharides having the respective masses estimated based on
the saccharide compositions are shown in Table 20.
TABLE 20 Composition of oligosaccharide Molecular Sulfate Peak no.
weight Fucose group Sodium 3-(1) 1564 5 8 8 1914 6 10 10 2016 6 11
11 2162 7 11 11 2264 7 12 12 2410 8 12 12 3-(2) 3110 10 16 16 3360
11 17 17 3462 11 18 18 3710 12 19 19 4308 14 22 22
It was shown that the sulfated fucan oligosaccharide fraction 3
contained various sulfated fucan oligosaccharides shown in the
table above.
Example 7
Preparation of Sulfated Fucan Oligosaccharide using Sulfated
Fucan-Digesting Enzyme, and Purification, Structural Analysis and
Physiological Activity Thereof
(1) In addition to the four distinct peaks observed in
DEAE-Cellulofine column chromatography as described in Example
2-(2), a broad peak eluted with a higher salt concentration was
observed. It was collected, designated as an oligosaccharide 4 and
subjected to NMR analysis as described in Example 2. As a result, a
spectrum almost identical to that for the sulfated fucan
oligosaccharide 1-(2) was observed. These results strongly
suggested that the oligosaccharide 4 had a structure in which
several molecules of the oligosaccharide 1-(2) were connected each
other. Then, the oligosaccharide 4 was digested using the sulfated
fucan-digesting enzyme as described in Example 1, and the digestion
product was analyzed using HPLC. The majority of the reaction
products was eluted at the same position as that for the sulfated
fucan oligosaccharide 1-(2).
The molecular weight of the oligosaccharide 4 as determined by gel
filtration using pullulan as a standard substance was shown to be
about triple of the oligosaccharide 1-(2).
Precise analysis of the .sup.1 H-NMR spectrum for the
oligosaccharide 4 revealed that the repeat of seven saccharide
residues was bound at the 3-position of fucose F6 in formula (IV)
via an .alpha.-(1.fwdarw.3) bond.
A monomer to a pentamer of the sulfated saccharide represented by
general formula (I) (i.e., sulfated saccharides of general formula
(I) wherein n=1 to 5) were obtained from digestion products of a
sulfated polysaccharide according to the method as described above.
##STR16##
wherein R is H or SO.sub.3 H and at least one of Rs is SO.sub.3
H.
As described above, it was confirmed that a sulfated
fucan-containing polysaccharide obtained from a brown alga such as
Kjellmaniella crassifolia was converted into smaller molecules by
treating it using a sulfated fucan-digesting enzyme, and that a
sulfated polysaccharide containing a sulfated saccharide of the
general formula above as an essential component of the constituting
saccharide was then obtained. The average molecular weight of the
sulfated polysaccharide extracted at pH 6-8 at 95.degree. C. for
about 2 hour as determined by gel filtration was about 200,000.
(2) The physiological activities of the sulfated saccharide
obtained in (1) above were examined. As a result, an activity of
inducing HGF production as determined according to the method
described in Example 1-(2) in WO 00/62785 was observed for the
sulfated polysaccharide of formula (I). In addition, the sulfated
saccharides of formula (I) were shown to be very useful in
retaining moisture as determined according to the method described
in Examples 8 and 9 in WO 99/41288.
The present invention provides a method for producing a sulfated
fucan oligosaccharide having a varying molecular weight, which is
useful as a reagent for glycotechnology, using a sulfated
fucan-digesting enzyme. The present invention also provides various
sulfated fucan oligosaccharides of which the structures have been
determined.
# SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 1 <210>
SEQ ID NO 1 <211> LENGTH: 1506 <212> TYPE: DNA
<213> ORGANISM: Fucanobacter lyticus <400> SEQUENCE: 1
agagtttgat cctggctcag attgaacgct ggcggcaggc ttaacacatg ca #agtcgagc
60 ggaaacgaga atagcttgct attcggcgtc gagcggcgga cgggtgagta at
#gcttggga 120 atatgcctaa tggtggggga caacagttgg aaacgactgc
taataccgca ta #atgtctac 180 ggaccaaagg aggggattct tcggaacctt
tcgccatttg attagcccaa gt #gagattag 240 ctagtaggta aggtaatggc
ttacctaggc gacgatctct agctggtttg ag #aggatgat 300 cagccacact
gggactgaga cacggcccag actcctacgg gaggcagcag tg #gggaatat 360
tgcacaatgg gcgaaagcct gatgcagcca tgccgcgtgt gtgaagaagg cc #ttcgggtt
420 gtaaagcact ttcagcgagg aggaaagggt gtagattaat actctgcatc tg
#tgacgtta 480 ctcgcagaag aagcaccggc taacttcgtg ccagcagccg
cggtaatacg ag #gggtgcaa 540 gcgttaatcg gaattactgg gcgtaaagcg
tgcgtaggtg gtttgttaag ca #agatgtga 600 aagccccggg ctcaacctgg
gaactgcatt ttgaactggc aaactagagt tt #tgtagagg 660 gtagtggaat
ttccagtgta gcggtgaaat gcgtagagat tggaaggaac at #cagtggcg 720
aaggcggcta cctggacaga gactgacact gaggcacgaa agcgtgggga gc #aaacagga
780 ttagataccc tggtagtcca cgccgtaaac gatgtcaact agccgtctgt ag
#acttgatc 840 tgtgggtggc gtagctaacg cgctaagttg accgcctggg
gagtacggcc gc #aaggttaa 900 aactcaaatg aattgacggg ggcccgcaca
agcggtggag catgtggttt aa #ttcgatgc 960 aacgcgaaga accttaccat
cccttgacat cctactaagt tactagagat ag #tttcgtgc 1020 cttcgggaaa
gtagtgacag gtgctgcatg gctgtcgtca gctcgtgttg tg #aaatgttg 1080
ggttaagtcc cgcaacgagc gcaaccccta tccttatttg ctagcgcgta at #ggcgagaa
1140 ctctaaggag actgccggtg ataaaccgga ggaaggtggg gacgacgtca ag
#tcatcatg 1200 gcccttacgg gatgggctac acacgtgcta caatggcaag
tacagagggc ag #caataccg 1260 cgaggtggag cgaatcccac aaagcttgtc
gtagtccgga ttggagtctg ca #actcgact 1320 ccatgaagtc ggaatcgcta
gtaatcgtag atcagaatgc tacggtgaat ac #gttcccgg 1380 gccttgtaca
caccgcccgt cacaccatgg gagtgggttg caaaagaagt gg #ctagttta 1440
acccttcggg gaggacggtc accactttgt gattcatgac tggggtgaag tc #gtaacaag
1500 gtagcc # # # 1506
* * * * *